Nanopipette analysis of polymers

Abstract

The disclosure relates to devices and instruments for detecting and individually analyzing biomolecules, biomolecular complexes and biomolecules with ligands attached thereon.

Description

RELATED APPLICATION[0001]This application claims priority under 35 U.S.C. § 119 to U.S. provisional patent application, U.S. Ser. No. 62 / 029,382, filed Jul. 25, 2014, entitled “NANOPIPETTE ANALYSIS OF POLYMERS,” the entire contents of which are incorporated herein by reference.Sequence Listing Submission Via EFS-Web[0002]A computer readable text file, entitled “SequenceListing.txt,” created on or about Jul. 24, 2015 with a file size of about 1 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.BACKGROUND[0003]It is increasingly realized that single molecule analysis techniques provide a depth of analysis not possible with traditional ensemble molecular methods. Gel analysis, microarray analysis and other methods that are commonly used in biochemistry and molecular biology take averaged readings of thousands of molecules. The target molecules typically have to be purified in substantial quantities (e.g., proteins) or must be ...

AI Technical Summary

Benefits of technology

[0009]According to some aspects, the present disclosure solves a number of problems found in existing approaches. In some embodiments, the disclosure overcomes issues associated with a low rate of polymer reaching a pore or nanopipette. In some embodiments, methods are provided that involve locating pores to a position that is in sufficient proximity to sample molecules (e.g., polymers) rather than bringing the molecules in proximity to the pores. In some embodiments, methods provided herein are more flexible than conventional approaches, allowing analysis to be done in situ, for example.

[0010]In some embodiments, providing sample molecules in an array format makes them readily accessible to a nanopipette, minimizing lag time between measurements. In some embodiments, methods provided in the disclosure controls the rate of translocation of a polymer through a pore such that it is not too rapid for detection, by immobilizing one end of the polymer, then elongating the polymer and then translating the nanopipette along the longitudinal length of the polymer at a controllable speed. The stretching of the polymer from the surface, enables the random motion of the polymer to be substantially reduced enabling clearer identification of signals in the recordings to be made.

Problems solved by technology

Due to PCR bias, coverage across the genome is not even, which makes de novo assembly challenging for large complex genomes.

Moreover, it is not currently possible to sequence molecules from a single cell without amplification.

In the case of proteins, it is challenging to decipher the identity of molecules within a complex mixture unless they are at high abundance.

One disadvantage of this approach is that it is not possible to keep all the molecules in synchrony (or in phase) and as the number of cycles increases the errors accumulate.

While such sequencing of single molecules directly does not suffer from the phasing problem, it can be compromised by the photophysics of individual fluorophores.

While it is possible to sequence single molecules of nucleic acids, there are apparently no single molecule methods for sequencing proteins.

Moreover, there are no amplification methods for proteins and proteins must be relatively pure and not part of a highly complex mixture in order to be analyzed.

From the foregoing it is clear that although progress has been made, there are a number of deficiencies in the technologies that represent the state of the art.

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